Rabies is a major global health issue that kills approximately 40,000 to 70,000 people per year and over 10 million people receive post-exposure prophylaxis (PEP) after exposure to potentially infected animals. Cost and compliance issues have greatly hampered the effectiveness of current vaccines. To confound this issue, current vaccines are not effective at preventing disease from several newly identified rabies related viruses. In addition, live virus was recently discovered in a lot of vaccine that was supposed to contain inactivated rabies virus. Taken together, the development of novel pre- and post-exposure vaccines is necessary to combat this global health issue. Since pre-exposure vaccination is reserved only for those at-risk populations, such as laboratory workers and veterinarians, PEP is the world-wide standard for human rabies prevention. As such, the development of alternative PEPs is the goal of this application. Current PEPs are comprised of one dose of passive immunization (rabies immune globulin) along with five active immunizations with rabies vaccines. We hypothesize that replication-deficient RVs will make excellent rabies PEPs. They are able to induce potent innate and adaptive immune responses, are intrinsically safe and can be grown to high liters. Two specific Aims are proposed: First, replication-deficient RV-based vectors will be constructed that have either P or M genes deleted. P- or M-deleted RVs that express two identical copies of RV G will also be constructed, which induces apoptosis and enhances anti-RV immune responses. We will characterize the genetic stability, growth kinetics, and transgene expression of these new viral vectors in vitro. Second, a hallmark of RV infection that makes PEP feasible is the relatively long period between the time of exposure at the peripheral site and the time when RV infects the central nervous system. As such, a rapid humoral response is absolutely required for a successful PEP. We will investigate the kinetics of the induced immune response in mice using these new vectors. RV G protein ELISAs, virus neutralization assays, and flow cytometry analysis will be used to study the induced humoral response. T-helper responses, which are important for strong B-cell responses, will be evaluated by proliferation assays against RV G and N. The most promising vectors will be tested in a well-defined Syrian hamster model to determine whether protection is provided after infection with a pathogenic RV strain. The responses will be compared with a licensed inactivated rabies vaccine. The goal of this application is to develop safe and effective alternatives to the current human rabies post- exposure prophylaxis. The development of treatment that relies on only one to two doses of vaccine instead of six inoculations will greatly enhance the effectiveness of rabies virus prevention. [unreadable] [unreadable] [unreadable]